Effect of Curcumin-Loaded Mesoporous Silica Nanoparticles on the Head and Neck Cancer Cell Line, HN5
, Elaheh Dalir Abdolahinia 2,†, Mohammad Ali Ghavimi 3, Solmaz Maleki Dizaj 4,*, Michael Aschner 5, Luciano Saso 6 and Haroon Khan 7,*
Round 1
Reviewer 1 Report
The manuscript explains a well-designed work on “Effect of Curcumin-Loaded Mesoporous Silica Nanoparticles 2 on Head and Neck Cancer Cell line HN5”
The volume of work done is truly appreciable and in my view, the manuscript may be accepted for publication.
However, there are a few minor queries which need to be addressed.
• English must be revised throughout the manuscript
• In the introduction section, curcumin-loaded nanoparticles related previous report needs to be updated.
• The source of a cell line is not mentioned in the material section
• The crystallinity index is not calculated, if possible calculate the same and add in the relevant section
• How did curcumin loading on nanoparticles was confirmed
Author Response
The volume of work done is truly appreciable and in my view, the manuscript may be accepted for publication. However, there are a few minor queries which need to be addressed. • English must be revised throughout the manuscript Answer: Thanks for your comment. The English language was comprehensively checked and corrected. • In the introduction section, curcumin-loaded nanoparticles related previous report needs to be updated. Answer: The curcumin-loaded nanoparticles related previous report was updated in the introduction section. • The source of a cell line is not mentioned in the material section Answer: It was mentioned. • The crystallinity index is not calculated, if possible calculate the same and add in the relevant section Answer: It was mentioned in the discussion section. • How did curcumin loading on nanoparticles was confirmed Answer: We confirmed it using drug loading test and XRD pattern. Please see the discussion section.Reviewer 2 Report
The manuscript deals with the effects of nanoparticles loaded with curcumin on head and neck cancer cells.
The field of nanoparticles is relevant and of interest to the scientific community, especially as carrier of drug that could induce cell death or toxicity in cancer cells.
The manuscript is quite clear, but there are gaps in content that need to be filled.
The introduction is poor, it should be better presented.
Line 44-45: the sentence needs to be clarified better. The radiation is not in line with the subject of the manuscript. Chemotherapy has limitations not only for side effects and sometime is required. I suggest to explain better.
Results of studies on the effects of curcumin in solid tumors and head and neck cancer should be added.
It should be better explained why the use of nanoparticles could be more beneficial than the use of free curcumin. I suggest to add other studies on this topici f any.
In the Materials and methods the details of the cells used should be mentioned, it is an immortalized cell line, etc..?
The paragraph “detection of ROS” is not in the right place. Please move it in results and discussion.
Nothing is mentioned about the nanoparticles alone, not loaded that can also play an active role in mediating biological effects. Please consider this point.
A paragraph on statistical analysis is totally missing.
In the results:
It is not clear which is the control group.
The title of the results of real time PCR is missing.
The title and a comment of staining for the detection of ROS are missing.
The evaluation on detection of ROS is a qualitative analysis. Is it possible to perfom a quantitative one?
The discussion needs to be reviewed. Explain better the difference between MSNs, free curcumin and curcumin-MSNs. Add bibliographic data on the state of the art on both the effects of curcumin and the effects of nanoparticles in cancer cells
Line 213. Hep G2 cells?
Line 214 It is mentioned that MSNs had no toxic effect in HN5 cells. Where is it showed? If it is a consideration. Provide the graph.
Line 214-215. The citation is not appropriate.
Nothing mentioned and discussed about detection of ROS.
Line 238-239: in vivo model are not highlighted in this study.
The English language is appropriate and understandable.
The figures and tables are appropriate. A graph of MSNs is not shown.
The literature should be updated with most recent publications.
Minor findings:
Line 95: not “assess”, but “assessment”
Line 102: please change “Bcl-2, Bax” with “Bcl-2 and Bax”
Line 121: specify the manufacturing company in the details of the kit
Lines 131-133: Move into discussion
Please check IC50 and not IC50 in the whole text.
Line 147 please change “Shows” with “shows”
Author Response
The manuscript deals with the effects of nanoparticles loaded with curcumin on head and neck cancer cells.
The field of nanoparticles is relevant and of interest to the scientific community, especially as carrier of drug that could induce cell death or toxicity in cancer cells.
The manuscript is quite clear, but there are gaps in content that need to be filled.
The introduction is poor, it should be better presented.
Answer: introduction section was improved.
Line 44-45: the sentence needs to be clarified better. The radiation is not in line with the subject of the manuscript. Chemotherapy has limitations not only for side effects and sometime is required. I suggest to explain better.
Answer: The mentioned sections were corrected and improved.
Results of studies on the effects of curcumin in solid tumors and head and neck cancer should be added.
Answer: The results of studies on the effects of curcumin in solid tumors and head and neck cancer were added to discussion section.
It should be better explained why the use of nanoparticles could be more beneficial than the use of free curcumin. I suggest to add other studies on this topici f any.
Answer: The mentioned sections were added to introduction.
In the Materials and methods the details of the cells used should be mentioned, it is an immortalized cell line, etc..?
Answer: The head and neck cancer cells HN-5 were used in this study, which was obtained from the Pasteur Institute of Iran. It was added in the Materials and methods section.
The paragraph “detection of ROS” is not in the right place. Please move it in results and discussion.
Answer: It was corrected.
Nothing is mentioned about the nanoparticles alone, not loaded that can also play an active role in mediating biological effects. Please consider this point.
Answer: There was no significant change in MSNs group as carrier compared to the control group (p>0.05). Therefore, due to the lack of cytotoxic effect of MSNs, other tests were not performed to investigate gene expression and ROS.
A paragraph on statistical analysis is totally missing.
Answer: It was added.
In the results:
It is not clear which is the control group.
Answer: Thanks for your precise review. The control group was untreated HN5 cells, which was mentioned in the manuscript.
The title of the results of real time PCR is missing.
Answer: It was added.
The title and a comment of staining for the detection of ROS are missing.
Answer: It was added.
The evaluation on detection of ROS is a qualitative analysis. Is it possible to perfom a quantitative one?
Answer: The preparation of nanoparticles and all investigations of this study was completed during the Covid-19 pandemic. Unfortunately, we did not perform a qualitative analysis of ROS at that time, so it is not possible for us to perform a new test.
The discussion needs to be reviewed. Explain better the difference between MSNs, free curcumin and curcumin-MSNs.
Answer: It was explain more in discussion.
Add bibliographic data on the state of the art on both the effects of curcumin and the effects of nanoparticles in cancer cells
Answer: The mentioned sections were added to introduction.
Line 213. Hep G2 cells?
Answer: It was corrected.
Line 214 It is mentioned that MSNs had no toxic effect in HN5 cells. Where is it showed? If it is a consideration. Provide the graph.
Answer: Thanks for your comment. The graph of MSNs is present in our unpublished article so we can't present them in two articles.
Line 214-215. The citation is not appropriate.
Answer: It was corrected.
Nothing mentioned and discussed about detection of ROS.
Answer: It was discussed about detection of ROS.
Line 238-239: in vivo model are not highlighted in this study.
Answer: Thanks for your precise review. The mentioned sentence was corrected.
The English language is appropriate and understandable.
Answer: Thanks for your comment. The English language was comprehensively checked and corrected.
The figures and tables are appropriate. A graph of MSNs is not shown.
Answer: There was no significant change in MSNs treated group as carriers compared to the control group (p>0.05). The figure of MSNs is present in our unpublished article so we can't present them in two articles.
The literature should be updated with most recent publications.
Answer: The manuscript was updated with recent publications.
Minor findings:
Line 95: not “assess”, but “assessment”
Answer: It was corrected.
Line 102: please change “Bcl-2, Bax” with “Bcl-2 and Bax”
Answer: It was corrected.
Line 121: specify the manufacturing company in the details of the kit
Answer: It was added.
Lines 131-133: Move into discussion
Answer: Lines 131-133 were moved into the discussion.
Please check IC50 and not IC50 in the whole text.
Answer: All of them are corrected.
Line 147 please change “Shows” with “shows”
Answer: It was corrected.
Reviewer 3 Report
Dear colleagues,
"Effects of Curcumin-loaded Mesosporous Silica Nanoparticles on Head and Neck Cancer Cell line HN5" by Dr Sharifi et al. is a preclinical analysis probing the effect of Curcumin nanoparticles in HN5 cell lines; the Manuscript infers that Curcumin incubation could led to increased proapoptotic protein expression, even though not reaching statistic significance.
The article was particularly engaging to read: Authors precisely described each and every step of the experiments; they furthermore explained thought process from which the study was conducted and limitations intrinsically developed. Curcumin-based treatment is nevertheless away from being even remotely considered on human cases; their results, however, could promote and implement current knowledge.
the article had minor flaws, mostly in:
- English syntax and sentence structure (i.e. line 80-84; the first sentence could be more intelligible if reversed). This revision is mandatory
- Abbreviation: Explaining every abbreviation on first occurrence would improve overall readers' understanding (i.e. PBS). This revision is mandatory
- proteins: Elaborating more on cited proteins would improve overall readers' understanding (GADPDH as housekeeping gene, BAX, BCL): the target audience is intended as Molecular analysts and Oncologists, albeit these findings could also reach "less-mocelularly-qualified" specialists. This suggestion could be rejected by authors if reasonably argued.
- figures and descriptions were greatly appreciated. It was a pleasure to read and look at them: they were clear, easy to understand, and accurate.
Kind Regards
Author Response
Reviewer 3
Dear colleagues,
"Effects of Curcumin-loaded Mesosporous Silica Nanoparticles on Head and Neck Cancer Cell line HN5" by Dr Sharifi et al. is a preclinical analysis probing the effect of Curcumin nanoparticles in HN5 cell lines; the Manuscript infers that Curcumin incubation could led to increased proapoptotic protein expression, even though not reaching statistic significance.
The article was particularly engaging to read: Authors precisely described each and every step of the experiments; they furthermore explained thought process from which the study was conducted and limitations intrinsically developed. Curcumin-based treatment is nevertheless away from being even remotely considered on human cases; their results, however, could promote and implement current knowledge.
the article had minor flaws, mostly in:
- English syntax and sentence structure (i.e. line 80-84; the first sentence could be more intelligible if reversed). This revision is mandatory
Answer: It was corrected by Prof. Michael Aschner, a coauthor of the article.
- Abbreviation: Explaining every abbreviation on first occurrence would improve overall readers' understanding (i.e. PBS). This revision is mandatory
Answer: It was done.
- proteins: Elaborating more on cited proteins would improve overall readers' understanding (GADPDH as housekeeping gene, BAX, BCL): the target audience is intended as Molecular analysts and Oncologists, albeit these findings could also reach "less-mocelularly-qualified" specialists. This suggestion could be rejected by authors if reasonably argued.
Answer: It was done.
- figures and descriptions were greatly appreciated. It was a pleasure to read and look at them: they were clear, easy to understand, and accurate.
Answer: Thanks for your comments.
Reviewer 4 Report
EVALUATION
This manuscript describes the preparation, characterization, and in vitro anticancer activity against Head and Neck Cancer Cell Line HN5. The authors synthesized Cur-MSN by sonication, followed by nanoprecipitation method to incorporate curcumin. From the results and discussion, there are still several obscurities. Here are some major comments that need to be addressed.
Major comments.
1. We encourage the author to check the grammatical errors throughout the manuscript.
2. The authors prepared the curcumin-loaded Mesoporous Silica Nanoparticles (Cur-MSN) by the nanoprecipitation assisted sonication method and the resulting MSN in the shape of rods. However, this method is a general one, and the resulting particles are usually spherical in shape. Usually, to specifically form rod-shaped particles, a special method or modification of the method is required. Can the author explain how rod-shaped particles are generated by the method used by the author? In addition, how the author removed the unloaded curcumin?
3. The authors mentioned the size of rod-shaped MSN in diameter. However, the rod-shaped is not spherical. As a result, it has both width and length.
4. The authors mentioned the IC50 of the Cur-MSN is better than free drug (Cur). Therefore, in our opinion the authors need to examine the blank MSN.
5. In terms of cur-MSNs, which have been reported many times, and curcumin itself, has been reported for its anticancer activity against many types of cancer cell lines. What is the novelty of this current research?
Minor comments.
1. The abbreviation should only be mentioned after full name at first and then and after that only mention the abbreviation only.
2. The unit of IC50 is not µm but µM (Figure 4)
3. Figure 4, x-axis in the log concentration mode?
4. Figure 5A and B can be combined
5. The XRD test needs to compare the free Cur and the Cur-MSN.
Comments for author File: 
Comments.docx
Author Response
This manuscript describes the preparation, characterization, and in vitro anticancer activity against Head and Neck Cancer Cell Line HN5. The authors synthesized Cur-MSN by sonication, followed by nanoprecipitation method to incorporate curcumin. From the results and discussion, there are still several obscurities. Here are some major comments that need to be addressed.
Major comments.
- We encourage the author to check the grammatical errors throughout the manuscript.
Answer: Thanks for your comment. The English language was comprehensively checked and corrected by Prof. Dr. Michael Aschner.
- The authors prepared the curcumin-loaded Mesoporous Silica Nanoparticles (Cur-MSN) by the nanoprecipitation assisted sonication method and the resulting MSN in the shape of rods. However, this method is a general one, and the resulting particles are usually spherical in shape. Usually, to specifically form rod-shaped particles, a special method or modification of the method is required. Can the author explain how rod-shaped particles are generated by the method used by the author? In addition, how the author removed the unloaded curcumin?
Answer: The silica nanoparticles was purchased in that morphology. The sonication method was used to precipitate curcumin in the pores of silica nanoparticles. The unloaded curcumin was removed using washing with ethanol.
- The authors mentioned the size of rod-shaped MSN in diameter. However, the rod-shaped is not spherical. As a result, it has both width and length.
Answer: We mentioned it as the mean particle size (that is including both width and length).
- The authors mentioned the IC50 of the Cur-MSN is better than free drug (Cur). Therefore, in our opinion the authors need to examine the blank MSN.
Answer: There was no significant change in MSNs treated group as carrier compared to the control group (p>0.05). The figure of MSNs is present in our unpublished article so we can't present them in two articles.
- In terms of cur-MSNs, which have been reported many times, and curcumin itself, has been reported for its anticancer activity against many types of cancer cell lines. What is the novelty of this current research?
Answer: Curcumin-Loaded Mesoporous Silica Nanoparticles in this study was prepared with MCM-41 family, and which effect of this nanoparticle was evaluated on Head and Neck Cancer Cell line HN5 for the first time.
Minor comments.
- The abbreviation should only be mentioned after full name at first and then and after that only mention the abbreviation only.
Answer: All of them corrected.
- The unit of IC50 is not µm but µM (Figure 4)
Answer: It was corrected.
- Figure 4, x-axis in the log concentration mode?
Answer: IC50 is defined as half maximal inhibitory concentration on cell viability. Thus, a logistic model can determine the concentration corresponding to a response midway between the estimates of the lower and upper plateaus, named the turning point (Ref: https://0-doi-org.brum.beds.ac.uk/10.1002/pst.426)
- Figure 5A and B can be combined
Answer: The controls are different and combining them can be confusing.
- The XRD test needs to compare the free Cur and the Cur-MSN.
Answer: We improved the discussion section.
Regards
Prof. Dr. Haroon Khan
Round 2
Reviewer 2 Report
The manuscript has been improved and updated.
